Tumor suppressors often function to maintain cell proliferation control pathways in an inactive state by suppressing positively acting growth regulators. Such growth control pathways can be viewed as constellations of protein complexes that assemble into a series of inter-connected sub-networks. The Hippo pathway is a protein kinase driven phosphorylation cascade containing multiple tumor suppressors that control cell growth via negative regulation of two growth promoting transcription factors - YAP and TAZ - in response to cell-cell contact signals. Moreover, the Hippo pathway is deactivated in many cancer types, including breast, kidney, and liver cancers. While certain key protein complexes and post-translational modifications in the pathway are known, our understanding of the global architecture of the pathway and how the architecture and modification status is altered upon loss of upstream tumor suppressors (NF2 and SAV) is limited. More generally, our ability to provide a quantitative description of complex signaling pathways is limited, due to the lack of systematic and generally applicable methods for determining the stoichiometry and occupancy of post-translational modifications and sub-network assemblies. In this regard, the stoichiometry of key post-translational modification events often dictates whether a particular signaling threshold has been overcome. This proposal seeks to develop a global and quantitative picture of the architecture and modification status of the Hippo pathway, and provide a description of how the organization of the network is altered upon loss of upstream tumor suppressors (NF2 and SAV). This will be accomplished by merging existing quantitative proteomics platforms developed in our lab with emerging multiplex AQUA (Absolute Quantification) proteomics approaches, which will allow the stoichiometry of protein complexes and modifications to be determined across a large network on interacting complexes. Functional analysis of key interactions and modifications will serve to validate the pathway. In addition, multiplex AQUA will be used to interrogate Hippo signaling dynamics in mammary epithelial cells grown in 3-dimensional cell culture, which accurately models breast tumor initiation and formation. The constellation of changes revealed by this study may represent a common cancer network signature, such that when the pathway becomes configured in this state, uncontrolled proliferation ensues leading to cancer emergence. In the future, the methods developed during this study could be used to uncover the cancer network signature for any signaling pathway following genetic or small-molecule perturbation.